Antiviral drugs for treatment of arenavrus infection

ABSTRACT

Compounds, methods and pharmaceutical compositions for treating viral infections, by administering certain novel compounds in therapeutically effective amounts are disclosed. Methods for preparing the compounds and methods of using the compounds and pharmaceutical compositions thereof are also disclosed. In particular, the treatment and prophylaxis of viral infections such as caused by hemorrhagic fever viruses is disclosed, i.e., including but not limited to, Arenaviridae (Junin, Machupo, Guanarito, Sabia, Lassa, Tacaribe, Pichinde, and LCMV), Filoviridae (Ebola and Marburg viruses), Flaviviridae (yellow fever, Omsk hemorrhagic fever and Kyasanur Forest disease viruses), and Bunyaviridae (Rift Valley fever and Crimean-Congo hemorrhagic fever).

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.11/712,918, filed Mar. 2, 2007, now pending, which claims benefit ofU.S. provisional application Ser. No. 60/778,107, filed Mar. 2, 2006,each of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The research described herein was supported in part by funds from theU.S. Government (Grant no. 7R43A1056525 and NIH SBIR grant R44 AI056525)and the U.S. Government may therefore have certain rights in theinvention.

FIELD OF THE INVENTION

The use of benzimidazole derivatives and analogs, as well ascompositions containing the same, for the treatment or prophylaxis ofviral diseases associated with the arenavirus family such as Lassafever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, andVenezuelan hemorrhagic fever.

BACKGROUND OF THE INVENTION

Viral hemorrhagic fever is a serious illness characterized by extensivevascular damage and bleeding diathesis, fever, and multiple organinvolvement. Many different viruses can cause this syndrome, each withits own animal reservoir, mode of transmission, fatality rate, andclinical outcome in humans. These viruses are distributed throughoutfour virus families, the Arenaviridae, Bunyaviridae, Filoviridae, andFlaviviridae. Several of these viruses generate significant morbidityand mortality and can be highly infectious by aerosol dissemination,promoting concern about weaponization. In 1999, the Centers for DiseaseControl and Prevention (CDC) identified and categorized potentialbiological terrorism agents as part of a Congressional initiative toupgrade bioterrorism response capabilities. Filoviruses and arenaviruseswere designated as Category A, defined as those pathogens with thehighest potential impact on public health and safety, potential forlarge-scale dissemination, capability for civil disruption, and greatestunmet need for public health preparedness. The National Institute ofAllergy and Infectious Diseases (NIAID) has since expanded the CategoryA list by adding several hemorrhagic bunyaviruses and flaviviruses. Inaddition, the Working Group on Civilian Biodefense described severalhemorrhagic fever viruses, including Lassa, as those with the greatestrisk for use as biological weapons and recommended the pursuit of newantiviral therapies.

Prevention and treatment options for hemorrhagic fever viruses arelimited. With the exception of an effective vaccine for yellow fever, nolicensed vaccines or FDA-approved antiviral drugs are available.Intravenous ribavirin has been used with some success to treatarenaviruses and bunyaviruses, although its use has significantlimitations as indicated below. In addition, there have been recentreports of promising vaccines for Ebola and Lassa. Although a successfulvaccine could be a critical component of an effective biodefense, thetypical delay to onset of immunity, potential side-effects, cost, andlogistics associated with large-scale civilian vaccinations against alow-risk threat agent suggest that a comprehensive biodefense include aseparate rapid-response element. Thus there remains an urgent need todevelop safe and effective products to protect against potentialbiological attack.

Lassa fever virus is a member of the Arenaviridae family, a family ofenveloped RNA viruses. Arenavirus infection in rodents, the natural hostanimal, is usually chronic and asymptomatic. Several arenaviruses cancause severe hemorrhagic fever in humans, including Lassa, Machupo,Guanarito, and Junin viruses. Transmission to humans can result fromdirect contact with infected rodents or their habitat, throughaerosolized rodent secretions, or through contact with the body fluidsof an infected person. Although arenaviruses are found world-wide, mostof the viral species are geographically localized to a particularregion, reflecting the range of the specific rodent host involved. TheArenaviridae family contains a single genus (Arenavirus) that is dividedinto two major lineages based on phylogenetic and serologicalexamination. Lassa fever is a member of the Old World arenaviruses; theNew World arenaviruses can be further divided into three clades (A-C),one of which (clade B) contains several of the pathogenic, Category Ahemorrhagic fever viruses.

Lassa fever is endemic in West Africa, particularly the countries ofGuinea, Liberia, Sierra Leone, and Nigeria. Human infections areestimated at 100,000 to 500,000 per year. Initial symptoms of Lassafever appear about 10 days after exposure, and include fever, sorethroat, chest and back pain, cough, vomiting, diarrhea, conjunctivitis,facial swelling, proteinuria, and mucosal bleeding. Clinical diagnosisis often difficult due to the nonspecific nature of the symptoms. Infatal cases, continuing progression of symptoms leads to the onset ofshock. Among hospitalized patients, the mortality rate is 15-20%,although the fatality rate for some outbreaks has been reported higherthan 50%. Infectious virus can remain in the bodily fluids ofconvalescent patients for several weeks. Transient or permanent deafnessis common in survivors and appears to be just as frequent in mild orasymptomatic cases as it is in severe cases. Lassa fever is occasionallyimported into Europe and the U.S., most recently in 2004. The risk ofthe virus becoming endemic outside of West Africa appears low due to thenature of the rodent host. However, the combination of increased worldtravel and viral adaptation presents a finite possibility of a virus“jumping” into a new ecosystem. For example, West Nile virus wasintroduced into the New York City area in 1999 and is now endemic in theU.S.

A small trial conducted in Sierra Leone in the 1980s demonstrated thatmortality from Lassa fever can be reduced in high-risk patients bytreatment with intravenous ribavirin, a nucleoside analog that exhibitsnonspecific antiviral activity. Ribavirin has been shown to inhibitLassa fever viral RNA synthesis in vitro. Although of limitedavailability, intravenous ribavirin is available for compassionate useunder an investigational new drug protocol. It is also available in oralform for treating hepatitis C (in combination with interferon), althoughless is known about the efficacy of orally-administered ribavirin fortreating Lassa fever. As a nucleoside analog, ribavirin can interferewith DNA and RNA replication, and in fact teratogenicity and embryolethality have been seen in several animal species. It is thereforecontraindicated for pregnant patients (a pregnancy category X drug). Inaddition, it is associated with a dose-related hemolytic anemia;although the anemia is reversible, anemia-associated cardiac andpulmonary events occur in approximately 10% of hepatitis C patientsreceiving ribavirin-interferon therapy. Intravenous ribavirin isexpensive, and daily I.V. administration to a large civilian populationin an emergency would be a cumbersome approach. It is possible thatfurther study may eventually support the use of oral interferon, eitheralone or in combination with other antivirals, for treatment of Lassafever. Successful antiviral therapy often involves administering acombination of pharmaceuticals, such as the treatment of chronichepatitis C with interferon and ribavirin, and treatment of AIDS withhighly active antiretroviral therapy (HAART), a cocktail of threedifferent drugs. Because of the high mutation rate and the quasispeciesnature associated with viruses, treatment with compounds that act onmultiple, distinct targets can be more successful than treatment with asingle drug.

The arenavirus genome consists of two segments of single-stranded RNA,each of which codes for two genes in opposite orientations (referred toas ambisense). The larger of the two segments, the L RNA (7.2 kb),encodes the L and Z proteins. The L protein is the RNA-dependent RNApolymerase, and the Z protein is a small zinc-binding RING fingerprotein which is involved in virus budding. The S RNA (3.4 kb) encodesthe nucleoprotein (NP) and the envelope glycoprotein precursor (GPC).

The envelope glycoprotein is embedded in the lipid bilayer thatsurrounds the viral nucleocapsid. The characteristics of the arenavirusglycoprotein suggest that it can be classified as a Type I envelope,which is typified by influenza hemagglutinin and found also inretroviruses, paramyxoviruses, coronaviruses, and filoviruses. Type Ienvelopes function both to attach the virus to specific host cellreceptors and also to mediate fusion of the viral membrane with the hostmembrane, thereby depositing the viral genome inside the target cell.Cotranslational translocation of the envelope protein across themembrane of the endoplasmic reticulum is facilitated by an N-terminalsignal peptide that is subsequently removed by a signal peptidase.Post-translational proteolysis further processes the envelope into anN-terminal subunit (denoted GP1 for arenaviruses), which contains thereceptor binding determinants, and a C-terminal transmembrane subunit(GP2), which is capable of undergoing the dramatic conformationalrearrangements that are associated with membrane fusion. The twosubunits remain associated with one another and assemble into trimericcomplexes of this heterodimer. Mature envelope glycoproteins accumulateat the site of viral budding, such as the plasma membrane, and thus areembedded within the envelope that the virus acquires as viral buddingoccurs.

The signal peptide of the arenavirus glycoprotein is quite unusual; at58 amino acids in length, it is larger than most signal peptides. Inaddition, it remains associated with the envelope and with maturevirions, and appears to be important for the subsequent GP1-GP2processing. This processing is essential for envelope function and ismediated by the cellular subtilase SKI-1/S1P. The envelope glycoproteininteracts directly with the host cellular receptor to facilitate viralentry into the target cell. The receptor for Old World arenaviruses isα-dystroglycan, a major component of the dystrophin glycoproteincomplex. The New World arenaviruses appear to have diverged from thisreceptor, as only the clade C viruses use α-dystroglycan as a majorreceptor. The Category A hemorrhagic New World arenaviruses usetransferrin receptor 1 to mediate entry into the host cell.

What is needed in the art are new therapies and preventives for thetreatment of viral infections and associated diseases, such as caused byhemorrhagic fever viruses like Arenaviruses.

The following publications represent the state of the art. They areincorporated herein by reference in their entirety.

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SUMMARY OF THE INVENTION

Provided are compounds and compositions and/or methods for the treatmentand prophylaxis of viral infections, as well as diseases associated withviral infections in living hosts. In particular, provided are compoundsand compositions and/or methods for the treatment and prophylaxis ofhemorrhagic fever viruses, such as Arenaviruses.

In an embodiment, a method for the treatment or prophylaxis of a viralinfection or disease associated therewith, comprising administering in atherapeutically effective amount to a mammal in need thereof, a compoundof Formula I or a pharmaceutically acceptable salt thereof is provided.In another embodiment, a pharmaceutical composition that comprises apharmaceutically-effective amount of the compound or apharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier is provided. In addition, compoundsof Formula I, as well as pharmaceutically-acceptable salts thereof areprovided.

The compounds of Formula I are of the following general formula:

-   Wherein R¹ and R² are independently hydrogen, alkyl, alkenyl,    alkynyl, cycloalkyl, arylalkyl, aryl, acyl, arylacyl, hydroxy,    alkyloxy, alkylthio, amino, alkylamino, acetamido, halogen, cyano or    nitro;-   R³ is hydrogen, acyl, arylacyl or sulfonyl; and-   Ar¹ and A² are independently (un)substituted aryl or heteroaryl.

In an embodiment, the mammal being treated is a human. In particularembodiments, the disease being treated is caused by a viral infection,such as by an Arenavirus. The Arenavirus may be selected from the groupconsisting of Lassa, Junin, Machupo, Guanarito, Sabia, WhitewaterArroyo, Chapare, LCMV, LCMV-like viruses such as Dandenong, Tacaribe,and Pichinde.

Details of methods and formulations are more fully described below.Other objects and advantages of the present invention will becomeapparent from the following description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the structure and antiviral activity of ST-37 andST-193. (1A) Benzimidazole derivative ST-37 (left) was modified togenerate ST-193 (right). (1B) Inhibition of LASV GP- or VSVg-pseudotypedHIV infection with ST-37 or ST-193. Infectivity measured by luciferasereporter relative to controls with no compound. Each point is an averageof three replicates, with error bars designating standard deviation.

FIGS. 2A and 2B show domain swapping which demonstrates that theC-terminal third of GP2 determines sensitivity to ST-193. (2A) Schematicrepresentation of LASV-LCMV chimeric GPs. Predicted TMD and heptadrepeat domains (HR1 and HR2) are indicated. The two GPs are spliced 12a.a. N-terminal of the predicted TMD. (2B) Pseudotype infectivity as inFIG. 1 using the arenavirus GPs shown (representative experiment).Average IC₅₀s±SEM for these constructs (across at least fourexperiments) were: LASV, 0.0016±0.0003 μM; LCMV, 31±4 μM;LASV_(N)-LCMV_(C), 13.6±2.2 μM; and LCMV_(N)-LASV_(C), 0.0005±0.0003 μM.

FIGS. 3A and 3B show ST-193 sensitivity determinants. (3A) Schematicrepresentation of an arenavirus GP2 subunit and the sites of 193Rvariations. The TCRV amino acid sequence is shown for the regionindicated, with sites identified as determinants of ST-193 sensitivityindicated with raised letters and numbering (TCRV GP amino acidnumbering). Predicted TMD and heptad repeat domains (HR1 and HR2) areindicated (TMD is outlined in the sequence). (3B) Single amino acidvariations that reduce ST-193 sensitivity in TCRV GP. Resistance is thefold change in IC₅₀ as measured with TCRV GP-pseudotyped HIV, relativeto wild-type, and averaged across at least four independent experiments(each in triplicate).

FIG. 4 shows amino acid alignment of a portion of the arenavirus GP2subunit. Amino acid numbering as in FIG. 3. Residues identified asST-193 sensitivity determinants (FIG. 3) are in bold type, and thepredicted TMD is indicated. The highlighted residues at positions 421and 425 are sensitivity determinants for which some sequence divergenceis observed across the Arenaviridae family. Abbreviations and GenBankaccession numbers are as follows: LASV, Lassa fever strain Josiah(J04324); LCMV, lymphocytic choriomeningitis strain Armstrong 53b(AY847350); MACV, Machupo strain Carvallo (AY619643); JUNV, Junin strainMC2 (D10072); TCRV, Tacaribe strain TRVL 11598 (P31840); GTOV, Guanarito(NC_(—)005077); SABV, Sabia (NC_(—)006317); LATV, Latino strain Maru10924 (AF485259); PICV, Pichinde (U77602); PIRV, Pirital (NC_(—)005894);and WWAV, Whitewater Arroyo (AF228063). LASV and LCMV are classified asOld World arenaviruses, while New World arenaviruses are represented byPICV and PIRV (clade A); MACV, JUNV, TCRV, GTOV, and SABV (clade B); andLATV (clade C). WWAV is likely a recombinant of clades A and B.

DETAILED DESCRIPTION

Compounds which are useful for the treatment and prophylaxis of viralinfections, particularly arenaviral infections, including diseasesassociated with arenaviral infections in living hosts, are provided. Inparticular, provided are compounds and compositions and/or methods forthe treatment and prophylaxis of hemorrhagic fever viruses, such asArenaviruses. However, prior to providing further detail, the followingterms will first be defined.

DEFINITIONS

In accordance with this detailed description, the followingabbreviations and definitions apply. It must be noted that as usedherein, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

The publications discussed herein are provided solely for theirdisclosure. Nothing herein is to be construed as an admission regardingantedating the publications. Further, the dates of publication providedmay be different from the actual publication dates, which may need to beindependently confirmed.

Where a range of values is provided, it is understood that eachintervening value is encompassed. The upper and lower limits of thesesmaller ranges may independently be included in the smaller, subject toany specifically-excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention. Alsocontemplated are any values that fall within the cited ranges.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Any methods and materials similar or equivalent to thosedescribed herein can also be used in practice or testing. Allpublications mentioned herein are incorporated herein by reference todisclose and describe the methods and/or materials in connection withwhich the publications are cited.

By “patient” or “subject” is meant to include any mammal. A “mammal,”for purposes of treatment, refers to any animal classified as a mammal,including but not limited to, humans, experimental animals includingrats, mice, and guinea pigs, domestic and farm animals, and zoo, sports,or pet animals, such as dogs, horses, cats, cows, and the like.

The term “efficacy” as used herein in the context of a chronic dosageregime refers to the effectiveness of a particular treatment regime.Efficacy can be measured based on change of the course of the disease inresponse to an agent.

The term “success” as used herein in the context of a chronic treatmentregime refers to the effectiveness of a particular treatment regime.This includes a balance of efficacy, toxicity (e.g., side effects andpatient tolerance of a formulation or dosage unit), patient compliance,and the like. For a chronic administration regime to be considered“successful” it must balance different aspects of patient care andefficacy to produce a favorable patient outcome.

The terms “treating,” “treatment,” and the like are used herein to referto obtaining a desired pharmacological and physiological effect. Theeffect may be prophylactic in terms of preventing or partiallypreventing a disease, symptom, or condition thereof and/or may betherapeutic in terms of a partial or complete cure of a disease,condition, symptom, or adverse effect attributed to the disease. Theterm “treatment,” as used herein, covers any treatment of a disease in amammal, such as a human, and includes: (a) preventing the disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it, i.e., causing the clinical symptomsof the disease not to develop in a subject that may be predisposed tothe disease but does not yet experience or display symptoms of thedisease; (b) inhibiting the disease, i.e., arresting or reducing thedevelopment of the disease or its clinical symptoms; and (c) relievingthe disease, i.e., causing regression of the disease and/or its symptomsor conditions. Treating a patient's suffering from disease related topathological inflammation is contemplated. Preventing, inhibiting, orrelieving adverse effects attributed to pathological inflammation overlong periods of time and/or are such caused by the physiologicalresponses to inappropriate inflammation present in a biological systemover long periods of time are also contemplated.

As used herein, “acyl” refers to the groups H—C(O)—, alkyl-C(O)—,substituted alkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—,alkynyl-C(O)—, substituted alkynyl-C(O)-cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—,substituted heteroaryl-C(O), heterocyclic-C(O)—, and substitutedheterocyclic-C(O)— wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Alkylamino” refers to the group —NRR where each R is independentlyselected from the group consisting of hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, substituted heterocyclic and whereeach R is joined to form together with the nitrogen atom a heterocyclicor substituted heterocyclic ring wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Alkenyl” refers to alkenyl group preferably having from 2 to 10 carbonatoms and more preferably 2 to 6 carbon atoms and having at least 1 andpreferably from 1-2 sites of alkenyl unsaturation.

“Alkoxy” refers to the group “alkyl-O—” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy,sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

“Alkyl” refers to linear or branched alkyl groups having from 1 to 10carbon atoms, alternatively 1 to 6 carbon atoms. This term isexemplified by groups such as methyl, t-butyl, n-heptyl, octyl and thelike.

“Amino” refers to the group —NH₂.

“Aryl” or “Ar” refers to an unsaturated aromatic carbocyclic group offrom 6 to 14 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl) which condensedrings may or may not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one, and the like) provided that the point ofattachment is through an aromatic ring atom.

“Substituted aryl” refers to aryl groups which are substituted with from1 to 3 substituents selected from the group consisting of hydroxy, acyl,acylamino, thiocarbonylamino, acyloxy, alkyl, substituted alkyl, alkoxy,substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, amidino, alkylamidino, thioamidino, amino, aminoacyl,aminocarbonyloxy, aminocarbonylamino, aminothiocarbonylamino, aryl,substituted aryl, aryloxy, substituted aryloxy, cycloalkoxy, substitutedcycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy,substituted heterocyclyloxy, carboxyl, carboxylalkyl,carboxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-substitutedcycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl,carboxyl-substituted heteroaryl, carboxylheterocyclic,carboxyl-substituted heterocyclic, carboxylamido, cyano, thiol,thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl,thioheteroaryl, substituted thioheteroaryl, thiocycloalkyl, substitutedthiocycloalkyl, thioheterocyclic, substituted thioheterocyclic,cycloalkyl, substituted cycloalkyl, guanidino, guanidinosulfone, halo,nitro, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, —S(O)₂-alkyl, —S(O)₂-substitutedalkyl, —S(O)₂-cycloalkyl, —S(O)₂-substituted cycloalkyl, —S(O)₂-alkenyl,—S(O)₂-substituted alkenyl, —S(O)₂-aryl, —S(O)₂-substituted aryl,—S(O)₂-heteroaryl, —S(O)₂-substituted heteroaryl, —S(O)₂-heterocyclic,—S(O)₂-substituted heterocyclic, —OS(O)₂-alkyl, —OS(O)₂-substitutedalkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic, —OS(O)₂—substituted heterocyclic, —OS(O)₂—NRR where R is hydrogen, or alkyl,—NRS(O)₂— alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituentsindependently selected from the group consisting of alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic and amino groups on thesubstituted aryl blocked by conventional blocking groups such as Boc,Cbz, formyl, and the like or substituted with —SO₂NRR where R ishydrogen or alkyl.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 8 carbon atomshaving a single cyclic ring including, by way of example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl and the like. Excludedfrom this definition are multi-ring alkyl groups such as adamantanyl,etc.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

“Heteroaryl” refers to an aromatic carbocyclic group of from 2 to 10carbon atoms and 1 to 4 heteroatoms selected from the group consistingof oxygen, nitrogen and sulfur within the ring or oxides thereof. Suchheteroaryl groups can have a single ring (e.g., pyridyl or furyl) ormultiple condensed rings (e.g., indolizinyl or benzothienyl) wherein oneor more of the condensed rings may or may not be aromatic provided thatthe point of attachment is through an aromatic ring atom. Additionally,the heteroatoms of the heteroaryl group may be oxidized, i.e., to formpyridine N-oxides or 1,1-dioxo-1,2,5-thiadiazoles and the like.Additionally, the carbon atoms of the ring may be substituted with anoxo (═O). The term “heteroaryl having two nitrogen atoms in theheteroaryl, ring” refers to a heteroaryl group having two, and only two,nitrogen atoms in the heteroaryl ring and optionally containing 1 or 2other heteroatoms in the heteroaryl ring, such as oxygen or sulfur.

“Substituted heteroaryl” refers to heteroaryl groups which aresubstituted with from 1 to 3 substituents selected from the groupconsisting of hydroxy, acyl, acylamino, thiocarbonylamino, acyloxy,alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, amidino,alkylamidino, thioamidino, amino, aminoacyl, aminocarbonyloxy,aminocarbonylamino, aminothiocarbonylamino, aryl, substituted aryl,aryloxy, substituted aryloxy, cycloalkoxy, substituted cycloalkoxy,heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substitutedheterocyclyloxy, carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxyl-substituted heterocyclic,carboxylamido, cyano, thiol, thioalkyl, substituted thioalkyl, thioaryl,substituted thioaryl, thioheteroaryl, substituted thioheteroaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheterocyclic,substituted thioheterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone, halo, nitro, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —S(O)₂-alkyl, —S(O)₂-substituted alkyl,—S(O)₂-cycloalkyl, —S(O)₂-substituted cycloalkyl, —S(O)₂-alkenyl,—S(O)₂-substituted alkenyl, —S(O)₂-aryl, —S(O)₂-substituted aryl,—S(O)₂-heteroaryl, —S(O)₂-substituted heteroaryl, —S(O)₂-heterocyclic,—S(O)₂-substituted heterocyclic, —OS(O)₂-alkyl, —OS(O)₂-substitutedalkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂— heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR where R is hydrogen oralkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituentsindependently selected from the group consisting of alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic and amino groups on thesubstituted aryl blocked by conventional blocking groups such as Boc,Cbz, formyl, and the like or substituted with —SO₂NRR where R ishydrogen or alkyl.

“Sulfonyl” refers to the group —S(O)₂R where R is selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein.

“Optionally substituted” means that the recited group may beunsubstituted or the recited group may be substituted.

“Pharmaceutically-acceptable carrier” means a carrier that is useful inpreparing a pharmaceutical composition or formulation that is generallysafe, non-toxic, and neither biologically nor otherwise undesirable, andincludes a carrier that is acceptable for veterinary use as well ashuman pharmaceutical use. A pharmaceutically-acceptable carrier orexcipient includes both one or more than one of such carriers.

“Pharmaceutically-acceptable cation” refers to the cation of apharmaceutically-acceptable salt.

“Pharmaceutically-acceptable salt” refers to salts which retain thebiological effectiveness and properties of compounds which are notbiologically or otherwise undesirable. Pharmaceutically-acceptable saltsrefer to pharmaceutically-acceptable salts of the compounds, which saltsare derived from a variety of organic and inorganic counter ions wellknown in the art and include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe molecule contains a basic functionality, salts of organic orinorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group.

Examples of suitable amines include, by way of example only,isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine,tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine,purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and thelike. It should also be understood that other carboxylic acidderivatives would be useful, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

Pharmaceutically-acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

A compound may act as a pro-drug. Pro-drug means any compound whichreleases an active parent drug in vivo when such pro-drug isadministered to a mammalian subject. Pro-drugs are prepared by modifyingfunctional groups present in such a way that the modifications may becleaved in vivo to release the parent compound. Prodrugs includecompounds wherein a hydroxy, amino, or sulfhydryl group is bonded to anygroup that may be cleaved in vivo to regenerate the free hydroxyl,amino, or sulfhydryl group, respectively. Examples of prodrugs include,but are not limited to esters (e.g., acetate, formate, and benzoatederivatives), carbamates (e.g., N,N-dimethylamino-carbonyl) of hydroxyfunctional groups, and the like.

“Treating” or “treatment” of a disease includes:

-   -   (1) preventing the disease, i.e. causing the clinical symptoms        of the disease not to develop in a mammal that may be exposed to        or predisposed to the disease but does not yet experience or        display symptoms of the disease,    -   (2) inhibiting the disease, i.e., arresting or reducing the        development of the disease or its clinical symptoms, or    -   (3) relieving the disease, i.e., causing regression of the        disease or its clinical symptoms.

A “therapeutically-effective amount” means the amount of a compound orantibody that, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically-effective amount” will vary depending on the compound,the disease, and its severity and the age, weight, etc., of the mammalto be treated.

Provided are compounds and compositions and/or methods for the treatmentand prophylaxis of viral infections, as well as diseases associated withviral infections in living hosts. In particular, provided are compoundsand compositions and/or methods for the treatment and prophylaxis ofhemorrhagic fever viruses, such as Arenaviruses.

In an embodiment, a method for the treatment or prophylaxis of a viralinfection or disease associated therewith, comprising administering in atherapeutically effective amount to a mammal in need thereof, a compoundof Formula I or a pharmaceutically acceptable salt thereof is provided.In another embodiment, a pharmaceutical composition that comprises apharmaceutically-effective amount of the compound or apharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier is provided. In addition, compoundsof Formula I, as well as pharmaceutically-acceptable salts thereof areprovided.

The compounds of Formula I are of the following general formula:

-   Wherein R¹ and R² are independently hydrogen, alkyl, alkenyl,    alkynyl, cycloalkyl, arylalkyl, aryl, acyl, arylacyl, hydroxy,    alkyloxy, alkylthio, amino, alkylamino, acetamido, halogen, cyano or    nitro;-   R³ is hydrogen, acyl, arylacyl or sulfonyl; and-   Ar¹ and Ar² are independently (un)substituted aryl or heteroaryl

Exemplary compounds of Formula I are shown below in Table 1:

TABLE I Exemplary compounds of Formula I No. formula structure/name600037 C₂₂H₂₁N₃O₂

(4-methoxy-benzyl)-[1-(4-methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600137 C₂₃H₂₄N₄O

(4-dimethylamino- benzyl)-[1-(2-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine 600144 C₂₂H₂₂N₄

(4-dimethylamino-benzyl)-[1- phenyl-1H-benzimidazol-5-yl]- amine 600145C₂₁H₁₈N₃OBr

(4-bromo-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600146 C₂₂H₂₁N₃O₂

(2-methoxy-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600147 C₂₃H₂₃N₃O₂

(4-ethoxy-benzyl)-[1-(4-methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600148 C₂₂H₂₁N₃O₂

(2-methoxy-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600149 C₂₅H₂₁N₃O

[1-(4-methoxy-phenyl)-1H- benzimidazol-5-yl]-napthalen-1- ylmethyl-amine600153 C₂₁H₁₉N₃O₂

2-[[1-(4-methoxy-phenyl)-1H- benzimidazol-5-ylamino]- methyl]-phenol600169 C₂₂H₂₁N₃O

(4-methoxy-benzyl)-(1-p-tolyl- 1H-benzimidazol-5-yl)-amine 600170C₂₂H₁₈N₃OCl

(4-chloro-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600172 C₂₃H₂₃N₃O₃

(3,4-dimethoxy-benzyl)-[1-(4- methoxy-phenyl)-1H-benzimidazol-5-yl]-amine 600173 C₂₀H₁₆N₃Br

(4-bromo-benzyl)-(1-phenyl-1H- benzimidazol-5-yl)-amine 600179C₃₀H₂₉N₃O₄S

N-(4-ethoxy-benzyl)-N-[1-(2- methoxy-phenyl)-1H- benzimidazol-5-yl]-4-methylbenzenesulfonamide 600188 C₂₂H₂₁N₃O

[1-(4-methoxy-phenyl)-1H- benzimidazol-5-yl]-(4-methyl- benzyl)-amine600189 C₂₃H₂₃N₃O₃

(2,3-dimethoxy-benzyl)-[1-(4- methoxy-phenyl)-1H-benzimidazol-5-yl]-amine 600190 C₂₂H₂₁N₃O₂

(4-methoxy-benzyl)-[1-(2- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600191 C₂₂H₂₁N₃O₂

(3-methoxy-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600192 C₂₃H₂₃N₃O₃

(2,3-dimethoxy-benzyl)-[1-(2- methoxy-phenyl)-1H-benzimidazol-5-yl]-amine 600193 C₂₄H₂₅N₃O

(4-isopropyl-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600196 C₂₁H₁₉N₃O

(4-methoxy-benzyl)-(1-phenyl- 1H-benzimidazol-5-yl)-amine 600362C₂₆H₂₇N₃O₂

N-(4-isopropyl-benzyl)-N-[1-4- methoxy-phenyl)-1H-benzimidazol-5-yl]-acetamide 600363 C₃₁H₃₁N₃O₃S

N-(4-isopropyl-benzyl)-N-[1-(4- methoxy-phenyl)-1H-benzimidazol-5-yl]-4- methylbenzenesulfonamide

In an embodiment, the mammal being treated is a human. In particularembodiments, the disease being treated is caused by a viral infection,such as by an Arenavirus. The Arenavirus may be selected from the groupconsisting of Lassa, Junin, Machupo, Guanarito, Sabia, WhitewaterArroyo, Chapare, LCMV, LCMV-like viruses such as Dandenong, Tacaribe,and Pichinde.

Pharmaceutical Formulations of the Compounds

In general, compounds will be administered in atherapeutically-effective amount by any of the accepted modes ofadministration for these compounds. The compounds can be administered bya variety of routes, including, but not limited to, oral, parenteral(e.g., subcutaneous, subdural, intravenous, intramuscular, intrathecal,intraperitoneal, intracerebral, intraarterial, or intralesional routesof administration), topical, intranasal, localized (e.g., surgicalapplication or surgical suppository), rectal, and pulmonary (e.g.,aerosols, inhalation, or powder). Accordingly, these compounds areeffective as both injectable and oral compositions. The compounds can beadministered continuously by infusion or by bolus injection.

The actual amount of the compound, i.e., the active ingredient, willdepend on a number of factors, such as the severity of the disease,i.e., the condition or disease to be treated, age, and relative healthof the subject, the potency of the compound used, the route and form ofadministration, and other factors.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound used, thetherapeutically-effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range which includes the IC₅₀ (i.e.,the concentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The amount of the pharmaceutical composition administered to the patientwill vary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions are administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications. Anamount adequate to accomplish this is defined as“therapeutically-effective dose.” Amounts effective for this use willdepend on the disease condition being treated as well as by the judgmentof the attending clinician depending upon factors such as the severityof the inflammation, the age, weight, and general condition of thepatient, and the like.

The compositions administered to a patient are in the form of 24pharmaceutical compositions described supra. These compositions may besterilized by conventional sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile aqueous carrier prior to administration. It will be understoodthat use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of pharmaceutical salts.

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically- ortherapeutically-effective amount. The therapeutic dosage of thecompounds will vary according to, for example, the particular use forwhich the treatment is made, the manner of administration of thecompound, the health and condition of the patient, and the judgment ofthe prescribing physician. For example, for intravenous administration,the dose will typically be in the range of about 0.5 mg to about 100 mgper kilogram body weight. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.Typically, the clinician will administer the compound until a dosage isreached that achieves the desired effect.

When employed as pharmaceuticals, the compounds are usually administeredin the form of pharmaceutical compositions. Pharmaceutical compositionscontain as the active ingredient one or more of the compounds above,associated with one or more pharmaceutically-acceptable carriers orexcipients. The excipient employed is typically one suitable foradministration to human subjects or other mammals. In making thecompositions, the active ingredient is usually mixed with an excipient,diluted by an excipient, or enclosed within a carrier which can be inthe form of a capsule, sachet, paper or other container. When theexcipient serves as a diluent, it can be a solid, semi-solid, or liquidmaterial, which acts as a vehicle, carrier, or medium for the activeingredient. Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing, for example, up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g., about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained, or delayed-release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The quantity of active compound in the pharmaceutical composition andunit dosage form thereof may be varied or adjusted widely depending uponthe particular application, the manner or introduction, the potency ofthe particular compound, and the desired concentration. The term “unitdosage forms” refers to physically-discrete units suitable as unitarydosages for human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient.

The compound can be formulated for parenteral administration in asuitable inert carrier, such as a sterile physiological saline solution.The dose administered will be determined by route of administration.

Administration of therapeutic agents by intravenous formulation is wellknown in the pharmaceutical industry. An intravenous formulation shouldpossess certain qualities aside from being just a composition in whichthe therapeutic agent is soluble. For example, the formulation shouldpromote the overall stability of the active ingredient(s), also, themanufacture of the formulation should be cost-effective. All of thesefactors ultimately determine the overall success and usefulness of anintravenous formulation.

Other accessory additives that may be included in pharmaceuticalformulations and compounds as follow: solvents: ethanol, glycerol,propylene glycol; stabilizers: EDTA (ethylene diamine tetraacetic acid),citric acid; antimicrobial preservatives: benzyl alcohol, methylparaben, propyl paraben; buffering agents: citric acid/sodium citrate,potassium hydrogen tartrate, sodium hydrogen tartrate, aceticacid/sodium acetate, maleic acid/sodium maleate, sodium hydrogenphthalate, phosphoric acid/potassium dihydrogen phosphate, phosphoricacid/disodium hydrogen phosphate; and tonicity modifiers: sodiumchloride, mannitol, dextrose.

The presence of a buffer is necessary to maintain the aqueous pH in therange of from about 4 to about 8. The buffer system is generally amixture of a weak acid and a soluble salt thereof, e.g., sodiumcitrate/citric acid; or the monocation or dication salt of a dibasicacid, e.g., potassium hydrogen tartrate; sodium hydrogen tartrate,phosphoric acid/potassium dihydrogen phosphate, and phosphoricacid/disodium hydrogen phosphate.

The amount of buffer system used is dependent on (1) the desired pH; and(2) the amount of drug. Generally, the amount of buffer used is in a0.5:1 to 50:1 mole ratio of buffenalendronate (where the moles of bufferare taken as the combined moles of the buffer ingredients, e.g., sodiumcitrate and citric acid) of formulation to maintain a pH in the range of4 to 8 and generally, a 1:1 to 10:1 mole ratio of buffer (combined) todrug present is used.

A useful buffer is sodium citrate/citric acid in the range of 5 to 50 mgper ml, sodium citrate to 1 to 15 mg per ml. citric acid, sufficient tomaintain an aqueous pH of 4-6 of the composition.

The buffer agent may also be present to prevent the precipitation of thedrug through soluble metal complex formation with dissolved metal ions,e.g., Ca, Mg, Fe, Al, Ba, which may leach out of glass containers orrubber stoppers or be present in ordinary tap water. The agent may actas a competitive complexing agent with the drug and produce a solublemetal complex leading to the presence of undesirable particulates.

In addition, the presence of an agent, e.g., sodium chloride in anamount of about of 1-8 mg/ml, to adjust the tonicity to the same valueof human blood may be required to avoid the swelling or shrinkage oferythrocytes upon administration of the intravenous formulation leadingto undesirable side effects such as nausea or diarrhea and possibly toassociated blood disorders. In general, the tonicity of the formulationmatches that of human blood which is in the range of 282 to 288 mOsm/kg,and in general is 285 mOsm/kg, which is equivalent to the osmoticpressure corresponding to a 0.9% solution of sodium chloride.

An intravenous formulation can be administered by direct intravenousinjection, i.v. bolus, or can be administered by infusion by addition toan appropriate infusion solution such as 0.9% sodium chloride injectionor other compatible infusion solution.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 5 to about 100 mg, more usually about 10 toabout 30 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 2000 mg of the activeingredient.

The tablets or pills may be coated or otherwise compounded to provide adosage form affording the advantage of prolonged action. For example,the tablet or pill can comprise an inner dosage and an outer dosagecomponent, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer which serves toresist disintegration in the stomach and permit the inner component topass intact into the duodenum or to be delayed in release. A variety ofmaterials can be used for such enteric layers or coatings, suchmaterials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate.

The liquid forms in which the novel compositions may be incorporated foradministration orally or by injection include aqueous solutions suitablyflavored syrups, aqueous or oil suspensions, and flavored emulsions withedible oils such as cottonseed oil, sesame oil, coconut oil, or peanutoil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically-acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically-acceptable excipients as describedsupra. Compositions in pharmaceutically-acceptable solvents may benebulized by use of inert gases. Nebulized solutions may be breatheddirectly from the nebulizing device or the nebulizing device may beattached to a face masks tent, or intermittent positive pressurebreathing machine. Solution, suspension, or powder compositions may beadministered from devices which deliver the formulation in anappropriate manner.

The compounds can be administered in a sustained release form. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the compounds, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981)and Langer, Chem. Tech. 12: 98-105 (1982) or poly(vinyl alcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand gamma ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-556,1983), non-degradable ethylene-vinyl acetate (Langer et al., supra),degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (i.e., injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

The compounds can be administered in a sustained-release form, forexample a depot injection, implant preparation, or osmotic pump, whichcan be formulated in such a manner as to permit a sustained-release ofthe active ingredient. Implants for sustained-release formulations arewell-known in the art. Implants may be formulated as, including but notlimited to, microspheres, slabs, with biodegradable or non-biodegradablepolymers. For example, polymers of lactic acid and/or glycolic acid forman erodible polymer that is well-tolerated by the host.

Transdermal delivery devices (“patches”) may also be employed. Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds in controlled amounts. The construction anduse of transdermal patches for the delivery of pharmaceutical agents iswell known in the art. See, e.g., U.S. Pat. No. 5,023,252, issued Jun.11, 1991, herein incorporated by reference. Such patches may beconstructed for continuous, pulsatile, or on-demand delivery ofpharmaceutical agents.

Direct or indirect placement techniques may be used when it is desirableor necessary to introduce the pharmaceutical composition to the brain.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system used for the transport ofbiological factors to specific anatomical regions of the body isdescribed in U.S. Pat. No. 5,011,472, which is herein incorporated byreference.

Indirect techniques usually involve formulating the compositions toprovide for drug latentiation by the conversion of hydrophilic drugsinto lipid-soluble drugs. Latentiation is generally achieved throughblocking of the hydroxy, carbonyl, sulfate, and primary amine groupspresent on the drug to render the drug more lipid-soluble and amenableto transportation across the blood-brain barrier. Alternatively, thedelivery of hydrophilic drugs may be enhanced by intra-arterial infusionof hypertonic solutions which can transiently open the blood-brainbarrier.

In order to enhance serum half-life, the compounds may be encapsulated,introduced into the lumen of liposomes, prepared as a colloid, or otherconventional techniques may be employed which provide an extended serumhalf-life of the compounds. A variety of methods are available forpreparing liposomes, as described in, e.g., Szoka et al., U.S. Pat. Nos.4,235,871, 4,501,728 and 4,837,028 each of which is incorporated hereinby reference.

Pharmaceutical compositions are suitable for use in a variety of drugdelivery systems. Suitable formulations for use in the present inventionare found in Remington's Pharmaceutical Sciences, Mace PublishingCompany, Philadelphia, Pa., 17th ed. (1985).

The provided compounds and pharmaceutical compositions show biologicalactivity in treating and preventing viral infections and associateddiseases, and, accordingly, have utility in treating viral infectionsand associated diseases, such as Hemorrhagic fever viruses, in mammalsincluding humans.

Hemorrhagic fever viruses (HFVs) are RNA viruses that cause a variety ofdisease syndromes with similar clinical characteristics. HFVs that areof concern as potential biological weapons include but are not limitedto: Arenaviridae (Junin, Machupo, Guanarito, Sabia, and Lassa),Filoviridae (Ebola and Marburg viruses), Flaviviridae (yellow fever,Omsk hemorrhagic fever and Kyasanur Forest disease viruses), andBunyaviridae (Rift Valley fever and Crimean-Congo hemorrhagic fever).The naturally-occurring arenaviruses and potential engineeredarenaviruses are included in the Category A Pathogen list according tothe Centers for Disease Control and Prevention as being among thoseagents that have greatest potential for mass casualties.

Risk factors include: travel to Africa or Asia, handling of animalcarcasses, contact with infected animals or people, and/or arthropodbites. Arenaviruses are highly infectious after direct contact withinfected blood and/or bodily secretions. Humans usually become infectedthrough contact with infected rodents, the bite of an infectedarthropod, direct contact with animal carcasses, inhalation ofinfectious rodent excreta and/or injection of food contaminated withrodent excreta. The Tacaribe virus has been associated with bats.Airborne transmission of hemorrhagic fever is another mode.Person-to-person contact may also occur in some cases.

All of the hemorrhagic fevers exhibit similar clinical symptoms.However, in general the clinical manifestations are non-specific andvariable. The incubation period is approximately 7-14 days. The onset isgradual with fever and malaise, tachypnea, relative bradycardia,hypotension, circulatory shock, conjunctival infection, pharyngitis,lymphadenopathy, encephalitis, myalgia, back pain, headache anddizziness, as well as hyperesthesia of the skin. Some infected patientsmay not develop hemorrhagic manifestations.

Methods of diagnosis at specialized laboratories include antigendetection by antigen-capture enzyme-linked immunosorbent assay (ELISA),IgM antibody detection by antibody-capture enzyme-linked immunosorbentassay, reverse transcriptase polymerase chain reaction (RT-PCR), andviral isolation. Antigen detection (by enzyme-linked immunosorbentassay) and reverse transcriptase polymerase chain reaction are the mostuseful diagnostic techniques in the acute clinical setting. Viralisolation is of limited value because it requires a biosafety level 4(BSL-4) laboratory.

EXAMPLE 1 Synthesis of Compounds

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees Celsius, and pressure is at or nearatmospheric.

The compounds are readily prepared via several divergent syntheticroutes with the particular route selected relative to the ease ofcompound preparation, the commercial availability of starting materials,and the like.

The compounds can be prepared from readily-available starting materialsusing the following general methods and procedures. It will beappreciated that where process conditions (i.e., reaction temperatures,times, mole ratios of reactants, solvents, pressures, etc.) are given,other process conditions can also be used unless otherwise stated.Optimum reaction conditions may vary with the particular reactants orsolvent used, but such conditions can be determined by one skilled inthe art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Second Edition, Wiley, New York, 1991, and references citedtherein.

Furthermore, the compounds will typically contain one or more chiralcenters. Accordingly, if desired, such compounds can be prepared orisolated as pure stereoisomers, i.e., as individual enantiomers ordiastereomers, or as stereoisomer-enriched mixtures. All suchstereoisomers (and enriched mixtures) are included unless otherwiseindicated. Pure stereoisomers (or enriched mixtures) may be preparedusing, for example, optically-active starting materials orstereoselective reagents well-known in the art. Alternatively, racemicmixtures of such compounds can be separated using, for example, chiralcolumn chromatography, chiral resolving agents, and the like.

Unless otherwise indicated, the products are a mixture of R, Senantiomers. However, when a chiral product is desired, the chiralproduct can be obtained via purification techniques which separateenantiomers from a R, S mixture to provide for one or the otherstereoisomer. Such techniques are known in the art.

In another embodiment, the compounds can be provided as pro-drugs whichconvert (e.g., hydrolyze, metabolize, etc.) in vivo to a compound above.

In the examples below, if an abbreviation is not defined above, it hasits generally accepted meaning. Further, all temperatures are in degreesCelsius (unless otherwise indicated). The following Methods were used toprepare the compounds set forth below as indicated.

Step 1: To a solution of dinitrofluorobenzene (251 μl, 2 mmol) in THF (2ml) was added cesium carbonate (780 mg, 2.4 mmol) and aniline (H₂N—Ar¹,2 mmol). The mixture was heated to 48° C. overnight. The reaction wascooled to room temperature and filtered through a pre-packed 5 g silicacartridge and eluted with EtOAc (˜15 ml). The solvent was removed invacuo and the crude material was carried forward without purification.

Step 2: To a solution of crude starting material from step 1 in EtOAcwas added a scoop of 10% Pd/C (˜50 mg). The vial was sealed, flushedwith Argon, and then placed under H₂ balloon. The mixture was stirred atroom temperature overnight. The reaction mixture was filtered through apad of Celite and eluted with EtOAc. The solvent was removed in vacuoand crude material was carried forward without purification.

Step 3: The crude material from step 2 was suspended in 4N HCl (2 ml)and formic acid (0.5 ml). The mixture was heated to 100° C. for 1.5hours. The reaction was cooled to room temperature and 5 N NaOH wasadded to adjust pH to 13. The mixture was extracted with DCM (3×5 ml).The combined organic layers were dried over MgSO₄, filtered, and solventevaporated in vacuo to give the crude product that was carried forwardwithout purification.

Step 4: To a solution of crude starting material from step 3 in DCM (3ml) was added aldehyde (Ar²—CHO, 2 mmol) and Na(AcO)₃BH (630 mg, 3mmol). The reaction was stirred at room temperature for 1.5 hours (whenreaction was complete by TLC). The crude reaction mixture was filteredand loaded onto a 40 g RediSep silica-gel cartridge and eluted with agradient of EtOAc in hexanes to yield the final product. The identitywas confirmed by LC-MS and ¹H NMR and purity confirmed by HPLC.

Synthesis of(4-isopropyl-benzyl)-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine

The compound was synthesized according to the General Proceduredescribed above. ¹H NMR (300 MHz, CDCl₃): δ 7.94 (s, 1H), 7.39 (m, 4H),7.24 (m, 4H), 7.04 (m, 3H), 6.74 (dd, 1H), 4.38 (s, 2H), 3.90 (s, 3H),2.93 (septet, 1H), 1.27 (d, 6H). ¹³C NMR (75 MHz, CDCl₃): δ 159.04,147.92, 145.19, 144.95, 142.03, 136.80, 129.57, 127.71, 127.43, 126.69,125.31, 115.04, 112.73, 110.74, 101.83, 55.64, 48.99, 33.83, 24.05.

EXAMPLE 2 Formulation 1

Hard gelatin capsules containing the following ingredients are prepared:

Ingredient Quantity (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

EXAMPLE 3 Formulation 2

A tablet formula is prepared using the ingredients below:

Ingredient Quantity (mg/capsule) Active ingredient 25.0 Cellulose,microcrystalline 200.0 Collodial silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

EXAMPLE 4 Formulation 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Ingredient Weight % Active Ingredient 5 Lactose 95

The active mixture is mixed with the lactose and the mixture is added toa dry powder inhaling appliance.

EXAMPLE 5 Formulation 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows:

Ingredient Quantity (mg/capsule) Active Ingredient 30.0 mg Starch 45.0mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone 4.0 mg (as10% solution in water) Sodium Carboxymethyl starch 4.5 mg Magnesiumstearate 0.5 mg Talc 1.0 mg Total 120 mg

The active ingredient, starch, and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinyl-pyrrolidone is mixed with the resultant powders, which arethen passed through a 16 mesh U.S. sieve. The granules so produced aredried at 50° to 60° C. and passed through a 16 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 30 mesh U.S. sieve, are then added to the granules,which after mixing, are compressed on a tablet machine to yield tabletseach weighing 150 mg.

EXAMPLE 6 Formulation 5

Capsules, each containing 40 mg of medicament, are made as follows:

Ingredient Quantity (mg/capsule) Active Ingredient 40.0 mg Starch 109.0mg Magnesium stearate 1.0 mg Total 150.0 mg

The active ingredient, cellulose, starch, an magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 150 mg quantities.

EXAMPLE 7 Formulation 6

Suppositories, each containing 25 mg of active ingredient, are made asfollows:

Ingredient Amount Active Ingredient 25 mg Saturated fatty acidsglycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

EXAMPLE 8 Formulation 7

Suspensions, each containing 50 mg of medicament per 5.0 ml dose, aremade as follows:

Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodiumcarboxymethyl cellose (11%) Microcrystalline cellulose (89%) 500 mgSucrose 1.75 g Sodium benzoate 10.0 mg Flavor and color q.v. Purifiedwater to 5.0 ml

The medicament, sucrose, and xanthan gum are blended, passed through aNo. 10 mesh U.S. sieve, and then mixed with a previously made solutionof the microcrystalline cellulose and sodium carboxymethyl cellulose inwater. The sodium benzoate, flavor, and color are diluted with some ofthe water and added with stirring. Sufficient water is then added toproduce the required volume.

EXAMPLE 9 Formulation 8

Hard gelatin tablets, each containing 15 mg of active ingredient, aremade as follows:

Ingredient Quantity (mg/capsule) Active Ingredient 15.0 mg Starch 407.0mg Magnesium stearate 3.0 mg Total 425.0 mg

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 560 mg quantities.

EXAMPLE 10 Formulation 9

An intravenous formulation may be prepared as follows:

Ingredient (mg/capsule) Active Ingredient 250.0 mg Isotonic saline 1000ml

Therapeutic compound compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle orsimilar sharp instrument.

EXAMPLE 11 Formulation 10

A topical formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 1-10 g Emulsifying Wax 30 g LiquidParaffin 20 g White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

EXAMPLE 12 Formulation 11

An aerosol formulation may be prepared as follows: A solution of thecandidate compound in 0.5% sodium bicarbonate/saline (w/v) at aconcentration of 30.0 mg/mL is prepared using the following procedure:

Preparation of 0.5% Sodium Bicarbonate/Saline Stock Solution: 100.0 mL

Ingredient Gram/100.0 mL Final Concentration Sodium Bicarbonate 0.5 g0.5% Saline q.s. ad 100.0 mL q.s. ad 100%

Procedure:

1. Add 0.5 g sodium bicarbonate into a 100 mL volumetric flask.2. Add approximately 90.0 mL saline and sonicate until dissolved.3. Q.S. to 100.0 mL with saline and mix thoroughly.

Preparation of 30.0 mg/mL Candidate Compound: 10.0 mL

Ingredient Gram/10.0 mL Final Concentration Candidate Compound 0.300 g30.0 mg/mL 0.5% Sodium Bicarbonate/ q.s. ad 10.0 mL q.s ad 100% SalineStock Solution

Procedure:

1. Add 0.300 g of the candidate compound into a 10.0 mL volumetricflask.2. Add approximately 9.7 mL of 0.5% sodium bicarbonate/saline stocksolution.3. Sonicate until the candidate compound is completely dissolved.4. Q.S. to 10.0 mL with 0.5% sodium bicarbonate/saline stock solutionand mix.

EXAMPLE 13 Determining Antiviral Activity of Compounds of the Invention

Work with Lassa fever virus presents significant logistical and safetyissues due to the requirement for maximum laboratory containment(BSL-4). Therefore, surrogate assays for anti-Lassa fever virus activitywere developed that would be suitable for evaluating large numbers ofcompounds under less-restrictive BSL-2 laboratory conditions. One suchassay was developed to identify compounds that can block Lassa virusentry into the host cell. This assay uses only the envelope glycoproteinfrom Lassa fever virus, not the virus itself, and thus can safely beperformed under normal BSL-2 conditions. The viral entry step is anattractive target for the development of antiviral pharmaceuticals,because it is an essential component of every viral life cycle. Inaddition, the antiviral targets, the interaction between the viralenvelope and the host cell and subsequent structural rearrangement ofthe envelope, are specific to the virus. Thus, effective inhibitors areless likely to interfere with host processes.

Viral pseudotypes, which are generated by cotransfection of the Lassaenvelope and a replication-defective HIV provirus with a luciferasereporter, are used to assess Lassa envelope function. The provirus isengineered so that the HIV envelope is not expressed, and thusheterologous viral envelope proteins are acquired as budding viralparticles nonspecifically capture cell surface proteins. Pseudotypesprepared in this manner will infect cells via the heterologous envelopeand are commonly used to assay functions of the heterologous envelope(2, 9, 26, 32, 34). Infection is measured by the luciferase signalproduced from the integrated HIV reporter construct. The amount ofinfectious virus used to infect a cell culture line is directlyproportional, over several orders of magnitude, to theluciferase-mediated luminescence produced in the infected cells. Thisassay was the basis of a high-throughput screen for Lassa virus entryinhibitors, against which a library of some 400,000 small moleculecompounds was tested. Compounds that inhibited luciferase activity by atleast 75% were subjected to a secondary specificity counter-screen, inwhich a second pseudotype using the unrelated Ebola virus glycoproteinwas used as a specificity control. Compounds that inhibited both typesof pseudotypes are likely either toxic to the cells or target the HIVplatform, and were thus rejected. The remaining pool of compoundsmeeting these criteria (about 300-400) were further investigated forchemical tractability, potency, and selectivity.

Initially, the chemical structures of the hit compounds were examinedfor chemical tractability. A chemically tractable compound is defined asone that is synthetically accessible using reasonable chemicalmethodology, and which possesses chemically stable functionalities andpotential drug-like qualities. Hits that passed this medicinal chemistryfilter were evaluated for their potency. Compound potency was determinedby evaluating inhibitory activity across a broad range ofconcentrations. Nonlinear regression was used to generate best-fitinhibition curves and to calculate the 50% effective concentration(EC₅₀). The selectivity or specificity of a given compound is typicallyexpressed as a ratio of its cytotoxicity to its biological effect. Acell proliferation assay is used to calculate a 50% cytotoxicityconcentration (CC₅₀); the ratio of this value to the EC₅₀ is referred toas the therapeutic index (T.I.=CC₅₀/EC₅₀). Two types of assays have beenused to determine cytotoxicity, both of which are standard methods forquantitating the reductase activity produced in metabolically activecells (28). One is a calorimetric method that measures the reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), andthe other uses fluorimetry to measure the reduction of resazurin (AlamarBlue). Selectivity could be further characterized by assessing theinhibitory action against viruses pseudotyped with unrelated viralenvelopes. The EC₅₀ for hit compounds was determined for HIV pseudotypesbearing one of three different viral envelopes: Lassa, Ebola, andvesicular stomatitis virus (VSV). The ratio between EC₅₀s thus became aquantitative measure of compound specificity, and compounds with ratiosless than 80 were rejected.

Twenty-five quality Lassa hits were discovered in the pool of initialhits from the pseudotype screening, all with EC₅₀ values below 1.8 μM.Ten of these compounds had EC₅₀s below 100 nM.

Compound ST-600037 was identified as one of the most potent andselective compounds from within the pool of 25 quality hits, in both theviral pseudotype assay and the Lassa fever virus plaque reduction assay(see Table 2 below). Chemical analogs of this compound were obtainedfrom commercial vendors or were synthesized, and these analogs weretested as described in order to define the relationship between chemicalstructure and biological activity. Several of these analogs, inparticular ST-600193, displayed enhanced potency and selectivityrelative to ST-600037. In addition, ST-600193 is also a potent inhibitorof pseudotyped viral infection mediated by the envelopes of the NewWorld arenaviruses Guanarito (EC₅₀<1 nM) and Tacaribe (EC₅₀=4 nM),demonstrating that this compound series may have utility for thetreatment of arenavirus diseases other than Lassa fever.

TABLE 2 Antiviral Activity of Compounds of the Present Invention.Activity against Lassa GP- Activity vs. pseudotyped-virus LFV* EC₅₀ EC₅₀No. (μM) specificity^(†) (μM) T.I.** formula structure/name 600037 0.016900 <0.1 >400 C₂₂H₂₁N₃O₂

(4-methoxy-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600137 >12 unknown n.d. n.d. C₂₃H₂₄N₄O

(4-dimethylamino-benzyl)-[1-(2- methoxy-phenyl)-1H-benzimidazol-5-yl]-amine 600144 11 >1 n.d. n.d. C₂₂H₂₂N₄

(4-dimethylamino-benzyl)-[1- phenyl-1H-benzimidazol-5-yl]- amine 6001450.04 300 n.d. n.d. C₂₁H₁₈N₃OBr

(4-bromo-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600146 0.3 >40 n.d. n.d. C₂₂H₂₁N₃O₂

(2-methoxy-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600147 0.003 >4000 n.d. n.d. C₂₃H₂₃N₃O₂

(4-ethoxy-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600148 9 none n.d. n.d. C₂₂H₂₁N₃O₂

(2-methoxy-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600149 0.06 >200 n.d. n.d. C₂₅H₂₁N₃O

[1-(4-methoxy-phenyl)-1H- benzimidazol-5-yl]-napthalen-1- ylmethyl-amine600153 0.18 >60 n.d. n.d. C₂₁H₁₉N₃O₂

2-[[1-(4-methoxy-phenyl)-1H- benzimidazol-5-ylamino]- methyl]-phenol600169 0.02 >500 n.d. n.d. C₂₂H₂₁N₃O

(4-methoxy-benzyl)-(1-p-tolyl- 1H-benzimidazol-5-yl)-amine 6001700.04 >200 n.d. n.d. C₂₂H₁₈N₃OCl

(4-chloro-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600172 0.7 >10 n.d. n.d. C₂₃H₂₃N₃O₃

(3,4-dimethoxy-benzyl)-[1-(4- methoxy-phenyl)-1H-benzimidazol-5-yl]-amine 600173 0.4 >30 n.d. n.d. C₂₀H₁₆N₃Br

(4-bromo-benzyl)-(1-phenyl-1H- benzimidazol-5-yl)-amine 600179 0.11 >100n.d. n.d. C₃₀H₂₉N₃O₄S

N-(4-ethoxy-benzyl)-N-[1-(2- methoxy-phenyl)-1H- benzimidazol-5-yl]-4-methylbenzenesulfonamide 600188 0.003 >4000 n.d. n.d. C₂₂H₂₁N₃O

[1-(4-methoxy-phenyl)-1H- benzimidazol-5-yl]-(4-methyl- benzyl)-amine600189 0.1 100 n.d. n.d. C₂₃H₂₃N₃O₃

(2,3-dimethoxy-benzyl)-[1-(4- methoxy-phenyl)-1H-benzimidazol-5-yl]-amine 600190 2 3 n.d. n.d. C₂₂H₂₁N₃O₂

(4-methoxy-benzyl)-[1-(2- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600191 0.04 >300 n.d. n.d. C₂₂H₂₁N₃O₂

(3-methoxy-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600192 5 none n.d. n.d. C₂₃H₂₃N₃O₃

(2,3-dimethoxy-benzyl)-[1-(2- methoxy-phenyl)-1H-benzimidazol-5-yl]-amine 600193 0.0011 13,000 <0.1 >400 C₂₄H₂₅N₃O

(4-isopropyl-benzyl)-[1-(4- methoxy-phenyl)-1H- benzimidazol-5-yl]-amine600196 0.03 >400 n.d. n.d. C₂₁H₁₉N₃O

(4-methoxy-benzyl)-(1-phenyl- 1H-benzimidazol-5-yl)-amine 600362 0.4 40n.d. n.d. C₂₆H₂₇N₃O₂

N-(4-isopropyl-benzyl)-N-[1-4- methoxy-phenyl)-1H-benzimidazol-5-yl]-acetamide 600363 0.2 100 n.d n.d C₃₁H₃₁N₃O₃S

N-(4-isopropyl-benzyl)-N-[1-(4- methoxy-phenyl)-1H-benzimidazol-5-yl]-4- methylbenzenesulfonamide *Lassa fever virus plaquereduction (Josiah strain) performed under BSL-4 conditions ^(†)EC₅₀ratio calculated as (EC₅₀ for negative control [VSV or Ebola, whicheverwas lower])/(EC₅₀ for Lassa) **T.I. (therapeutic index) is the ratio ofcytotoxicity to effective anti-Lassa concentrations (CC₅₀/EC₅₀) on Verocells

EXAMPLE 14 Determining Antiviral Activity of Compounds of the Invention

As discussed in Example 13 above, anti-infective drug discovery for LASVpresents significant logistical and safety challenges due to therequirement for maximum laboratory containment (BSL-4). Therefore, asurrogate assay, in which the LASV envelope glycoprotein (GP) wasincorporated into retroviral pseudotypes, was used as a high-throughputscreening (HTS) platform. Arenavirus entry is mediated by this singlevirally-encoded protein, categorized as a class I viral fusion protein(Gallaher W R (2001) BMC Microbiol 1:1; York J (2005) Virology343:267-274; Eschli B (2006) J Virol 80-5897-5907), facilitating theeffective use of pseudotypes for antiviral screening. Inhibitors of LASVGP-mediated viral entry could thus be identified from a library of smallmolecule compounds. As an essential component of the viral life cycle,the entry process is an attractive target for the development ofantiviral pharmaceuticals. For example, two distinct classes of viralentry inhibitor, enfuvirtide (Matthews T, et al. (2004) Nat Rev DrugDiscov 3:215-225) and maraviroc (Dorr P, et al. (2005) Antimicrob AgentsChemother 49:4721-4732), have recently been approved for H IV treatment.

Materials and Methods

Virus, Cells, and Compounds. Viruses and Vero cells were describedpreviously (Bolken T C, et al. (2006) Antiviral Res 69:86-97). 293T/17cells (ATCC CRL-11268) were maintained in Dulbecco's modified Eaglemedium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS)(Invitrogen) at 37° C. with 5% CO₂. Initial compound lots were purchasedfrom commercial suppliers (ST-37 from Asinex and ST-193 fromInterBioScreen), while subsequent batches have been custom synthesizedby a number of different vendors. Compound stock solutions were made at10 mM in dimethyl sulfoxide (DMSO).

Viral Glycoprotein Cloning. Viral RNA isolated with a QIAamp Viral RNAkit (Qiagen) served as a template for cDNA synthesis using SuperScriptOne-Step RT-PCR (Invitrogen) and GP-specific primers. GP inserts, exceptfor JUNV GP (see below), were subsequently subcloned into the mammalianexpression vector pCAGGS (Niwa H, et al. (1991) Gene 108:193-199). JuninGP cDNA was subcloned into pCI (Promega) via PCR with engineered,flanking Kpn I-Bam HI restriction sites. GenBank accession numbers areprovided in the FIG. 4 legend, with the following deviations relative tothe deposited sequences (all numbering relative to the relevant GP ORF):MACV GP had one synonymous nucleotide (nt) substitution (T₅₅₂C); JUNV GPhad one synonymous nt substitution (C₁₄₄₆T); PICV GP had one codingsubstitution (G₃₉₅A nt change coding for a S₁₃₂N a.a. change); and TCRVGP differed from a previously reported sequence (Allison LMC, et al.(1991) J Gen Virol 72:2025-2029) at three locations: C₂₉₇T (synonymoussubstitution) and GA to AG at nt 1336-7, coding for a E₄₄₆R a.a. change.The LASV-LCMV chimeras were created by overlapping PCR, fusing the 5′1245 (LASV) or 1263 nt (LCMV) with the 3′ 231 (LASV) or 234 nt (LCMV) ofthe GP ORF (numbering includes termination codon); the protein fusionjunction is thus C-terminal of a common TEML (single a.a. code)sequence. Arenavirus GP point mutations were introduced with theQuikChange Site-Directed Mutagenesis kit (Stratagene). LASV V₄₃₁M (LASV#1 in Table 4) was generated by substitution of 2 nt (G₁₂₉₁A, T₁₂₉₃G)within codon 431, LASV V₄₃₅M (LASV #2 in Table 4) was a single ntsubstitution (G₁₃₀₃A), and LASV dbl contained both a.a. changes. LCMVM₄₃₇V (LCMV #1 in Table 4) was generated by a A₁₃₀₉G nt substitution,and LCMV dbl (M₄₃₇V, M₄₄₁V) also contained a A₁₃₂₁G nt change. PichindeT₄₄₅V (Pichinde #1 in Table 4) was made by substituting GT for AC at nt1333-4.

Generation of ST-193-resistant TCRV variants. Vero cells were initiallyinfected with TCRV at an MOI of 0.1 in minimal essential media (MEM)with 2% FBS in the presence of 1.2 μM ST-193. Virus was harvested whencytopathic effect became apparent (˜7-10 days) and passaged again at ahigher ST-193 concentration (1.8 μM); this process was repeated at 2.4and 3 μM ST-193. RNA was isolated from the last virus harvest and cDNAprepared as above, followed by cloning into TOPO TA vector pCR2.1(Invitrogen). Multiple clones were then sequenced and subcloned intopCAGGS using Eco RI-Xho I restriction sites.

Pseudotyped virus production. Pseudovirions were generated with athree-plasmid, HIV-based expression system (Naldini L, et al. (1996)Science 272-263-267). 293T/17 cells were transfected (CalPhos MammalianTransfection kit, BD Biosciences) with a 1:1:1.7 ratio of pΔR8.2,pHR′-Luc, and GP expression construct (1:1:0.6 for VSVg) and inducedwith 10 mM sodium butyrate for 6 hours, 20-26 hours post-transfection(applied only to non-VSVg envelopes). Supernatants were harvested 48hours post-transfection, clarified by low-speed centrifugation (100×gfor 4 min.), filtered with a 0.45 μM syringe filter, and stored inaliquots at −80° C.

Infections and inhibition assays. 293T/17 cells seeded inpoly-D-lysine-coated 96-well plates were infected with pseudovirions inDMEM+7.5% FBS+0.5% DMSO and assayed for firefly luciferase activity(Luciferase Assay System from Promega) at 72 hours post-infection.Luminescense was quantitated on a Wallac EnVision 2102 Multilabel platereader (PerkinElmer) using 1 second read time per well. Luciferasesignal was directly proportional to inoculum size over several orders ofmagnitude. In order to test for antiviral activity, serial compounddilutions in DMSO were added in triplicate to cells immediately prior tovirus addition, maintaining 0.5% final DMSO concentration in all wells.Each 96-well plate had a minimum of four replicates of a negativecontrol (no virus) and eight replicates of a positive control (viruswithout compound). Test well luciferase activity was converted to % ofthe positive control, and IC₅₀s were calculated using XLfit (IDBS) forMicrosoft Excel with a one site dose-response curve fit.

Identification and activity of ST-193. A chemically diverse library ofabout 400,000 small molecules was screened with retroviral-basedpseudotypes incorporating the LASV GP. Hit compounds were filteredthrough a battery of follow-up tests, including specificity assays,cytotoxicity assays, confirmation assays against authentic Lassa fevervirus, and validation of antiviral activity with re-synthesizedcompound. ST-37, a benzimidazole derivative exhibiting an average IC₅₀against LASV GP pseudotypes of 16 nM (FIG. 1A), was identified from thisprocess. In order to characterize the relationship between chemicalstructure and biological activity (structure-activity relationship, orSAR), analogs of ST-37 were assayed for antiviral activity. One of theseanalogs, ST-193 (FIG. 1A;(4-isopropyl-benzyl)-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine),substitutes an isopropyl for a methoxy group, resulting in significantlygreater antiviral potency (FIG. 1B). A non-arenavirus envelope, the Gprotein from vesicular stomatitis virus (VSVg), is used as a specificitycontrol. As a practical consideration, compounds are tested atconcentrations no higher than 50 μM, a level at which ST-193 exhibitsvisual evidence of insolubility (crystals observed by light microscopy);this is also near the 50% cytotoxicity concentration (ST-193 CC₅₀=48 μMon 293T/17 cells), and thus the VSVg inhibition seen at the highestconcentration is considered to be nonspecific activity (IC₅₀ around 30μM; FIG. 1B).

The activity and specificity of ST-193 has been assessed by a variety ofassays, including pseudotype infectivity, virus yield reduction, plaquereduction, and cytopathic effect. The pseudotype platform was the mostreproducible and amenable to direct comparison between diverse viruses,particularly given that several viruses of interest are restricted touse under BSL-4 containment. As shown in Table 3, ST-193 potentlyinhibits envelopes derived from clade B New World arenaviruses, thephylogenetic cluster containing all four South American hemorrhagicfever viruses (Junin, Machupo, Guanarito, and Sabia). ST-193 alsopotently inhibits viral entry mediated by the GP from the prototypic NewWorld arenavirus, Tacaribe virus (TCRV). TCRV, another clade B member,is not known to be a significant human pathogen. Surprisingly, ST-193exhibits only a nonspecific level of activity against the LASV-relatedLCMV GP, with an IC₅₀ some four orders of magnitude greater than againstLASV (Table 3).

TABLE 3 ST-193 antiviral activity against retroviral-based pseudotypesincorporating heterologous envelope glycoproteins. GP Phylogeny of GPsource IC₅₀ (μM) ± SEM Lassa Old World arenavirus; Josiah strain 0.0016± 0.0003 LCMV Old World arenavirus; Armstrong 53b 31 ± 4  Pichinde NewWorld arenavirus, clade A 2.6 ± 0.5 Junín New World arenavirus, clade B10.0002 ± 0.00003 Machupo New World arenavirus, clade B1 0.0023 ± 0.0013Tacaribe New World arenavirus, clade B1 0.004 ± 0.002 Guanarito NewWorld arenavirus, clade B2 0.00034 ± 0.00007 Sabia New World arenavirus,clade B3 0.012 ± 0.003 VSV rhabdovirus  29 ± 2.5

Mapping determinants of ST-193 sensitivity. To gain insight into themechanism of ST-193 inhibition and potential binding sites, threestrategies were employed to identify determinants of ST-193 sensitivitywithin the arenavirus envelope glycoprotein. First, domain swappingexperiments were performed using two closely-related arenavirus GPs, onesensitive (LASV) and one insensitive (LCMV) to ST-193. Second, anonpathogenic surrogate virus (TCRV) was passaged in the presence ofST-193 to select for less-sensitive variants. Finally, site-directedmutagenesis identified two significant amino acids located within thepredicted transmembrane domain (TMD) of the GP2 subunit.

C-terminal portion of the LASV GP2 subunit confers ST-193 sensitivity.Retroviral pseudotypes incorporating chimeric arenavirus envelopes wereconstructed to identify the region dictating ST-193 sensitivity.Constructs that exchanged the C-terminal one-third of the GP2 subunit(76 a.a. of LASV, 77 a.a. of LCMV) were found to retain viral entryfunction. These chimeras partition the entire endodomain and predictedTMD from the signal peptide, the GP1 subunit, and most of the GP2subunit ectodomain (FIG. 2A), including the N-terminal and most of theC-terminal heptad repeats characteristic of class I viral fusionproteins (Gallaher W R, et al. (2001) BMC Microbiol 1:1.16-18; York J,et al. (2005) Virology 343:267-274; and Eschli B, et al. (2006) J Virol80:5897-5907). ST-193 sensitivity was found to be conferred by theC-terminal portion of GP2 (FIG. 2B).

Generation and characterization of ST-193-resistant TCRV variants. Inorder to genetically map sensitivity determinants, TCRV was seriallypassaged in the presence of escalating concentrations of ST-193 toselect for variants with decreased sensitivity. A selected population(193R) was thus generated that yielded equivalent viral titers whencultured in the presence or absence of 3 μM ST-193; in contrast,unselected TCRV titer is reduced 1000-fold under correspondingconditions (data not shown). RNA from the selected population was usedto synthesize cDNA, and multiple GP sequences were determined fromclones derived from this cDNA. Coding changes found within any GPsequence were subsequently introduced into a TCRV GP expression plasmidfor viral pseudotype production in order to directly evaluate thecontribution of a given mutation to ST-193 sensitivity. Although thisapproach gives little consideration to viral fitness, it allows forsimple and rapid identification of multiple sensitivity determinants.

Individual 193R mutations that reduce ST-193 sensitivity were found inor immediately N-terminal of the predicted TMD of the GP2 subunit (FIG.3). The 193R variations resulted in a range of resistance as singleamino acid changes increased the ST-193 IC₅₀ by 30-fold to more than1000-fold. At position 413, located within the predicted ectodomain nearthe TMD, two distinct mutations (Q₄₁₃H and Q₄₁₃R) resulted in greatlyreduced ST-193 sensitivity.

Interestingly, resistance to a previously-described inhibitor of NewWorld arenavirus entry, ST-294, has been mapped to a similar locationwithin the TCRV GP2 subunit (Bolken T C, et al. (2006) Antiviral Res69:86-97). Three of the four recognized ST-294-resistant TCRV variants(DR1-4) also exhibited reduced sensitivity to ST-193 (FIG. 3), while thefourth (DR3, or S₄₃₃I) retained full sensitivity. The ST-294 DR4mutation (F₄₃₆I) was also identified from within the 193R population.Conversely, the other 193R variants retain ST-294 sensitivity (data notshown). A mutation identified from a third distinct TCRV selection,R₄₁₂T, was also found to display ST-193 resistance (FIG. 3). This thirdscreen was designed to identify resistance to ST-761, another New Worldarenavirus inhibitor that targets the GP protein. As with the ST-294comparison, 193R variants retain sensitivity to ST-761, with theexception of the F₄₃₆I variant. ST-193, ST-294, and ST-761 arechemically diverse small molecules with no obvious commoncharacteristic.

Two TMD residues regulate ST-193 sensitivity. Alignment of arenavirus GPprotein sequences reveals conservation of the majority of ST-193sensitivity determinants identified above (FIG. 4). Positions 421 and425 show some diversity, however, which could in part explain therelative insensitivity of LCMV and Pichinde virus GPs to ST-193.Position 421 in particular is striking in that arenavirus GPs sensitiveto ST-193 in the nanomolar range contain valine, while those that arenot contain other residues (methionine or threonine). To directly testthe functional significance of residue 421 with respect to ST-193sensitivity, the LASV GP was engineered to contain the correspondingamino acid from ST-193-insensitive LCMV at one or both of these sites(Table 4). Additionally, some of the reciprocal changes were made in theLCMV GP, and the Val₄₂₁ present in ST-193-sensitive GPs was introducedinto the Pichinde GP. Viral pseudotypes were made to evaluate ST-193sensitivity for each of these genotypes.

TABLE 4 Site-directed mutagenesis of predicted ST-193 sensitivitydeterminants. Site #1 Site #2 IC₅₀ relative to GP (421)* (425)* IC₅₀(μM) ± SEM LASV LASV wt Val Val 0.0016 ± 0.0003 - 1 - LASV dbl Met Met24 ± 3  15,200 LASV #1 Met Val 1.4 ± 0.3 850 LASV #2 Val Met 6.1 ± 1.33800 LCMV wt Met Met 31 ± 4  19,600 LCMV #1 Val Met 27 ± 4  16,900 LCMVdbl Val Val 26 ± 4  16,200 Pichinde wt Thr Phe 2.6 ± 0.5 1600 Pichinde#1 Val Phe 0.046 ± 0.025 29 *TCRV GP numbering of aligned arenavirus GPsequences

Introduction of a methionine into either position 421 or 425dramatically reduces the sensitivity of LASV to ST-193, and substitutionof both residues further reduces the sensitivity such that LASV dbl GP(V₄₂₁M, V₄₂₅M) and LCMV GP display nearly equivalent ST-193 sensitivity(Table 4). The reciprocal substitutions (LCMV dbl and LCMV #1), however,do not convert LCMV GP to greater sensitivity, indicating that otherresidues play a role in LCMV resistance to ST-193 inhibition. Thesignificance of position 421 is further highlighted by the increasedST-193 sensitivity of Pichinde GP when valine is exchanged for thenative threonine residue (Table 4).

The benzimidazole derivative described here, ST-193, has beendemonstrated to be a potent inhibitor of arenavirus entry. Without beingbound by theory, the present evidence suggest that ST-193 does notdirectly block virus attachment. First, domain swapping experimentsdemonstrate that the difference in sensitivity between LASV and LCMVresides not within the receptor-binding GP1 domain, but in the GP2domain. Second, no sensitivity determinants were identified within GP1following selection for ST-193-resistant arenavirus. Finally, arenavirusentry is mediated by a diversity of host cell surface receptors that donot appear to correspond to ST-193 sensitivity. LASV and LCMV utilizeα-dystroglycan (Cao W, et al. (1998) Science 282:2079-2081), while theCategory A hemorrhagic New World arenaviruses use transferrin receptor 1(Radoshitzky S R, et al. (2007) Nature 446:92-96). Clade C New Worldarenaviruses, like the Old World arenaviruses, use α-dystroglycan(Spiropoulou C F, et al. (2002) J Virol 76:5140-5146). Some other NewWorld arenaviruses, including ST-193-sensitive TCRV, likely use adistinct, yet-unidentified receptor (Flanagan M L, et al. (2008) J Virol82:938-948; Oldenburg J, et al. (2007) Virology 364:132-139; Reignier T,et al. (2008) Virology 371:439-446; and Rojek J M, et al. (2006)Virology 349:476-491). These observations, combined with the presence ofmultiple ST-193 sensitivity determinants within the fusogenic GP2subunit, suggest that ST-193 blocks GP-mediated entry at apost-attachment stage.

ST-193 sensitivity is modulated by a 28 amino acid span of the GP2subunit of the envelope protein, encompassing nearly the entirepredicted TMD. Although these results implicate this region in ST-193activity, the location and spacing of the identified sensitivitydeterminants suggest that at least some of these sites may not bedirectly involved in inhibitor binding. Notably, the two residuesimplicated in the phylogeny of ST-193 sensitivity, positions 421 and425, are located four amino acids apart. Within an α-helical TMD, thesetwo residues are likely adjacent to one another; position 418, anotherimportant sensitivity determinant (FIG. 2B), lies on the same helicalface as well. The predicted physical property of ST-193 (averagecalculated partition coefficient, or cLogP, of 5.5) is consistent withbinding a hydrophobic pocket. ST-193 might prevent or perturbconformational rearrangement of GP, or inhibit lipid mixing, therebypreventing fusion with the host cell. Alternatively, ST-193 couldinteract with the membrane-proximal domain, in the 409-413 regionidentified following genetic selection; although this region is morehydrophilic, the membrane-proximal ectodomain of HIV gp41 (the class Iviral fusion protein equivalent of arenavirus GP2) is a target ofbroadly-neutralizing monoclonal antibodies (Sun Z-Y J, et al. (2008Immunity 28:52-63). Another possibility is that ST-193 might disruptprotein-protein interactions within or near the TMD. Interactionsbetween the TMDs of alphavirus E1 and E2 proteins are important forfusion (Sjöberg M, et al. (2003) J Virol 77:3441-3450). Several recentstudies have investigated the unusual role of the arenavirus GP signalpeptide (Agnihothram S S, et al. (2006) J Virol 80:5189-5198;Agnihothram S S, et al. (2007) J Virol 81:4331-4337; Eichler R, et al.(2003) EMBO Rep 4:1084-1088; Eichler R, et al. (2004) J Biol Chem279:12293-12299; Froeschke M, et al. (2003) J Biol Chem 278:41914-41920;Kunz S, et al. (2003) Virology 314:168-178; Saunders A A, et al. (2007)J Virol 81:5649-5657; Schrempf S, et al. (2007) J Virol 81: 12515-12524;York J, et al. (2006) J Virol 80:7775-7780; York J, et al. (2007) JVirol 81:13385-13391; York J, et al. (2004) J Virol 78:10783-10792),which remains associated with GP and is required for efficient GP1-GP2processing, transport to the plasma membrane, membrane fusion, and virusinfectivity. This cleaved, 58 amino acid peptide has two conservedhydrophobic domains and a hydrophilic amino terminus. The interactionbetween the stable signal peptide and the processed GP1-GP2 complex,then, might provide a vulnerable antiviral target specific toarenaviruses. Sensitivity to at least three dissimilar antiviralcompounds (ST-193, ST-294, and ST-761) has been mapped to the TMD regionof GP2, with overlapping yet quite distinct patterns of geneticresistance. By way of analogy, several different classes of smallmolecule HIV entry inhibitor, including maraviroc, are thought to bindwithin a pocket created by four TMDs of CCR5, an important HIVco-receptor (Kondru R (2008) Mol Pharmacol 73:789-800).

Arenaviruses constitute an important class of hemorrhagic feverpathogen, with five viruses designated Category A. The potency andspectrum of ST-193 activity make it a strong antiviral compound for theprevention and treatment of disease caused by these viruses.

All references cited herein are herein incorporated by reference intheir entirety for all purposes.

1. A method for the treatment or prophylaxis of an Arenavirus viralinfection or disease associated therewith, comprising administering in atherapeutically effective amount to a mammal in need thereof, a compoundof Formula I below:

wherein R¹ and R² are independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, arylalkyl, aryl, acyl, arylacyl, hydroxy, alkyloxy,alkylthio, amino, alkylamino, acetamido, halogen, cyano or nitro; R³ ishydrogen, acyl, arylacyl or sulfonyl; and Ar¹ and Ar² are independently(un)substituted aryl or heteroaryl, wherein said Arenavirus is selectedfrom the group consisting of Lassa, Junin, Machupo, Guanarito, Sabia,Whitewater Arroyo, Chapare, LCMV and LCMV-like viruses.
 2. The method ofclaim 1, wherein R¹ is hydrogen.
 3. The method of claim 1, wherein R² ishydrogen.
 4. The method of claim 1, wherein R³ is hydrogen.
 5. Themethod of claim 1, wherein R³ is —S(O)₂-substituted aryl.
 6. The methodof claim 5, wherein R³ is —S(O)₂-substituted phenyl.
 7. The method ofclaim 6, wherein R³ is —S(O)₂-alkoxyphenyl.
 8. The method of claim 7,wherein R³ is —S(O)₂-methoxyphenyl.
 9. The method of claim 7, wherein R³is —S(O)₂-p-methoxyphenyl.
 10. The method of claim 1, wherein Ar¹ isunsubstituted aryl.
 11. The method of claim 10, wherein Ar¹ isunsubstituted phenyl.
 12. The method of claim 1, wherein Ar¹ ismono-substituted aryl.
 13. The method of claim 12, wherein Ar¹ ismono-substituted phenyl.
 14. The method of claim 13, wherein Ar¹ isalkoxyphenyl.
 15. The method of claim 14, wherein Ar¹ is methoxyphenyl.16. The method of claim 15, wherein Ar¹ is o-methoxyphenyl.
 17. Themethod of claim 15, wherein Ar¹ is p-methoxyphenyl.
 18. The method ofclaim 1, wherein Ar² is substituted aryl.
 19. The method of claim 18,wherein Ar² is substituted phenyl.
 20. The method of claim 19, whereinAr² is mono-substituted phenyl.
 21. The method of claim 20, wherein Ar²is alkoxyphenyl.
 22. The method of claim 21, wherein Ar² ismethoxyphenyl.
 23. The method of claim 22, wherein Ar² iso-methoxyphenyl.
 24. The method of claim 22, wherein Ar² ism-methoxyphenyl.
 25. The method of claim 22, wherein Ar² isp-methoxyphenyl.
 26. The method of claim 21, wherein Ar² isethoxyphenyl.
 27. The method of claim 26, wherein Ar² is p-ethoxyphenyl.28. The method of claim 20, wherein Ar² is alkylphenyl.
 29. The methodof claim 28, wherein Ar² is methylphenyl.
 30. The method of claim 29,wherein Ar² is p-methylphenyl.
 31. The method of claim 20, wherein Ar²is propylphenyl.
 32. The method of claim 31, wherein Ar² isp-propylphenyl.
 33. The method of claim 32, wherein Ar² isp-isopropylphenyl.
 34. The method of claim 13, wherein Ar² ishalo-substituted phenyl.
 35. The method of claim 34, wherein Ar² isp-halo-substituted phenyl.
 36. The method of claim 35, wherein Ar² isp-bromophenyl.
 37. The method of claim 36, wherein Ar² isp-chlorophenyl.
 38. The method of claim 13, wherein Ar² ishydroxyphenyl.
 39. The method of claim 38, wherein Ar² iso-hydroxyphenyl.
 40. The method of claim 13, wherein Ar² isdimethylaminophenyl.
 41. The method of claim 40, wherein Ar² isp-dimethylaminophenyl.
 42. The method of claim 12, wherein Ar² is—S(O)₂-substituted aryl.
 43. The method of claim 42, wherein Ar² is—S(O)₂-substituted phenyl.
 44. The method of claim 43, wherein Ar² is—S(O)₂-alkoxyphenyl.
 45. The method of claim 44, wherein Ar² is—S(O)₂-methoxyphenyl.
 46. The method of claim 45, wherein Ar² is—S(O)₂-p-methoxyphenyl.
 47. The method of claim 20, wherein Ar² isdiphenyl.
 48. The method of claim 1, wherein compound of Formula I isselected from the group consisting of(4-Methoxy-benzyl)-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(4-Dimethylamino-benzyl)-[1-(2-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(4-Dimethylamino-benzyl)-(1-phenyl-1H-benzimidazol-5-yl)-amine,(4-Dimethylamino-benzyl)-(1-phenyl-1H-benzimidazol-5-yl)-amine,(4-Bromo-benzyl)-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(2-Methoxy-benzyl)-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(4-Ethoxy-benzyl)-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(2-Methoxy-benzyl)-[1-(2-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,[1-(4-Methoxy-phenyl)-1H-benzimidazol-5-yl]-naphthalen-1-ylmethyl-amine,(4-Methoxy-benzyl)-(1-p-tolyl-1H-benzimidazol-5-yl)-amine,(4-Chloro-benzyl)-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(3,4-Dimethoxy-benzyl)-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(4-Bromo-benzyl)-(1-phenyl-1H-benzimidazol-5-yl)-amine,N-(4-Ethoxy-benzyl)-N-[1-(2-methoxy-phenyl)-1H-benzimidazol-5-yl]-4-methylbenzenesulfonamide,[1-(4-Methoxy-phenyl)-1H-benzimidazol-5-yl]-(4-methyl-benzyl)-amine,(2,3-Dimethoxy-benzyl)-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(4-Methoxy-benzyl)-[1-(2-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(3-Methoxy-benzyl)-[1-(4-methoxy-phenyl)-1H benzimidazol-5-yl]-amine,(2,3-Dimethoxy-benzyl)-[1-(2-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(4-Isopropyl-benzyl)-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-amine,(4-Methoxy-benzyl)-(1-phenyl-1H-benzimidazol-5-yl)-amine,N-(4-Isopropyl-benzyl)-N-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-acetamide,andN-(4-Isopropyl-benzyl)-N-[1-(4-methoxy-phenyl)-1H-benzimidazol-5-yl]-4-methylbenzenesulfonamide.49. The method of claim 1, wherein the mammal is a human.
 50. The methodof claim 1, which further comprises co-administering a combination ofcidofovir, cyclic cidofovir, or salts, esters, or prodrugs thereof. 51.The method of claim 1, wherein said Arenavirus is Junin.
 52. The methodof claim 1, wherein said Arenavirus is Machupo.
 53. The method of claim1, wherein said Arenavirus is Guanarito.
 54. The method of claim 1,wherein said Arenavirus is Sabia.
 55. The method of claim 1, whereinsaid Arenavirus is Lassa.
 56. The method of claim 1, wherein saidArenavirus is Tacaribe.
 57. The method of claim 1, wherein saidArenavirus is Pichinde.
 58. The method of claim 1, wherein saidLCMV-like viruses are selected from the group consisting of Dandenong,Tacaribe, and Pichinde.